The bDNA assay developed by Chiron Diagnostics and now owned by Bayer Diagnostics uses linear, rather than exponential, signal amplification to increase the sensitivity and specificity of quantitative hybridization in diagnostic tests. Collins et al., NUCLEIC ACID RESEARCH 25(15):2979-2984 (1997). The bDNA assay is used to quantify RNA and DNA targets from a variety of sources. The sensitivity and specificity of the assay are derived in part through the judicious choice of oligonucleotide probes that constitute the probe set.
In one bDNA assay, capture probes (“CPs”) are attached to a solid support and capture extender (“CE”) probes attach to the CPs to mediate the capture of DNA or RNA targets to the solid support. The DNA or RNA targets are labeled using a large number (typically>30) target-specific oligonucleotides called label extender (“LE”) probes, which mediate the hybridization of bDNA amplifier molecules to the CEs. Hybridization of the targets to the solid support is detected typically by way of alkaline phosphatase probes, which are bound to the branches of the bDNA. The signal amplification is the product of the bound LE probes, the number of branches on the bDNA amplifier molecule, and the number of alkaline phosphatase binding sites on each branch of the bDNA molecules. This type of bDNA assay, referred to as the “first generation” bDNA assay or the “Quantiplex” bDNA assay, quantifies between 10,000 and 10,000,000 molecules/mL and has been used for the detection of HIV, HBV, and HCV. Collins et al., supra at 2979.
In the “second generation” bDNA assay, also called the “Enhanced Sensitivity” or “ES” bDNA assay, the LE probes bind preamplifiers, which in turn bind numerous bDNA amplifier molecules. The result of the second generation bDNA assays is stronger signal amplification and lower detection limits. For example, the second generation bDNA assay was able to quantify 500 molecules/mL of HIV RNA. See, Kern et al., JOURNAL OF CLINICAL MICROBIOLOGY 34(12):3196-3202 (1996).
A negative side effect of the first and second generation bDNA assays is background noise attributed to non-specific hybridization of the amplification sequences to non-target molecules. For example, non-specific hybridization of any of the following may result in an increase in background noise: hybridization of the bDNA to the CE probe (rather than the LE probe) and non-specific hybridization of the CE probe to the LE probe (rather than the CE probe to the target and the target to the LE probe). Non-specific hybridization in the bDNA assay has the effect of reducing the sensitivity of the assay.
To reduce non-specific hybridization in the bDNA assay, the non-natural bases, isoguanosine (“iso-G”) and isocytidine (“iso-C”), both of which have no detectable interaction with any of the natural DNA or RNA bases, have been incorporated into the amplification sequences. Iso-G and iso-C form standard Watson and Crick interactions with each other; however, because the hydrogen bonding pattern between the iso-G and iso-C is different from the hydrogen bonding pattern between the natural bases, there is no interaction between iso-G and iso-C and the natural bases. See, U.S. Pat. No. 5,681,702. Sequences having iso-G/iso-C base pairs are −2° C. more stable than their G/C congeners.
The incorporation of iso-G and iso-C bases into the second generation bDNA assay is known as the “third generation” bDNA assay or the “System 8” bDNA assay procedure. See, Collins et al., supra, and U.S. Pat. No. 5,681,702. In the third generation bDNA assay, preferably every third or fourth nucleotide of the capture, preamplifier, amplifier, and/or label probes are either iso-G or iso-C, both of which base pair with each other, but not with natural bases. With the incorporation of the iso-G and iso-C nucleotides into the bDNA assay, background noise resulting from non-specific hybridization is reduced and signal amplification is increased. Control over non-specific hybridization with iso-G and iso-C allows for the use of larger LE preamplifier probes, larger bDNA amplifier molecules, or more layers of amplification to improve the sensitivity of the bDNA assay, since signal can be augmented without equal amplification of noise. For example, Collins et al. document that detection of 5 attomol of oligonucleotide target with alkaline phosphatase probe has a signal to noise (“S/N”) ratio of 5.5 whereas two-layered amplification has an S/N ratio of 19.6 and three-layered amplification has an S/N ratio of 154.3. Using the third generation bDNA assay, Collins et al. was able to quantify 60 molecules/mL of HIV RNA. Collins et al., supra at 1982.
The second and third generation bDNA assays have use in a multiplex format. See, Collins et al., supra at 2983. In multiplex assays, many targets are analyzed simultaneously. Multiplex assays have been used to genotype single nucleotide polymorphisms (“SNPs”), i.e., single point variations in genomic DNA, and to screen for various cytokines in a sample. See, Iannone et al., CYTOMETRY 39:131-140 (2000); Collins et al., supra at 2983; and de Jager et al., CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY 10(1):133-139 (2003).
SNPs represent the most abundant form of a genetic variation and occur, on average, at every 1-2 kb in the human genome. Over 4 million SNPs have been identified (see, www.ncbi.nlm.nih.gov/SNP), and of these SNPs over 1.2 million have been mapped to the human genome (see, snp.cshl.org). Due to the abundant repetitive sequences in the human genome, all of the SNPs contained in the databases are not necessarily true polymorphisms or they may not be polymorphic in a specific population of interest. The identification of SNPs has uses beyond human diagnostic analyses. For example, the identification of SNPs is useful for genetic mapping, genetic diversity analyses, and marker-assisted breeding in a wide-variety of species including, plants, mammals, and micro-organisms. Accordingly, accurate and efficient genotyping is a prerequisite to the identification of SNPs. See, Lindroos et al., NUCLEIC ACID RESEARCH 30(14):1-9 (2002).
Cytokines are soluble proteins that are secreted by cells of the immune system. These proteins can alter the behavior and properties of different cell types. Different cytokines possess biological overlapping functions and therefore, have the ability to regulate the production of other cytokines. Thus, analysis of the function of a complete set of cytokines within a microenvironment, i.e., a site of inflammation, is frequently of more value than analysis of a single isolated cytokine. Cytokines can be quantitated at various levels. Multiplex assays for detection of cytokines at the messenger RNA (“mRNA”) and cellular levels have limitations, such as the need for large volumes of sample or detection of a precursor protein rather than a native protein. See, de Jager et al., supra.
In order to use the bDNA assay in a multiplex format, multiple CP and CE sequences must be used in order to attach multiple targets to the solid support. Collins et al., supra. The problem with using the six-base code second and third generation bDNA assays in multiplex format is the potential 3-mer cross-hybridization that may occur between the natural bases of the multiple targets and the capture, preamplifier, amplifier, and label probes. More specifically, as mentioned above, the second and third generation bDNA assays both have a non-natural base in every fourth nucleotide position of the capture, preamplifier, amplifier, and/or label probes; accordingly, under this design, three of four potential natural bases are positioned between two non-natural bases in each of the probes. Since each natural base has only one successful match, hybridization of a natural base with any one of its three mismatched bases will result in a significant decrease in the efficacy of the multiplex bDNA assay. Further, as complicated multiplex assays are developed, unwanted cross-reactivity between analytes will be difficult to trouble-shoot and remove.
Accordingly, there remains a need in the art to design and generate multiple highly specific sequences for use in bDNA singleplex and multiplex assays that do not cross-hybridize. The present invention addresses this need in the art by designing and generating highly orthogonal six-code universal sequences that minimize or eliminate the 3-mer cross-hybridization inherent in the second and third generation bDNA assays as they are presently known in the art. By developing universal sequences with little or no cross-reactivity, the accuracy of bDNA assays for the detection and screening of viruses, retroviruses, SNPs, cytokines, and gene amplifications and deletions, is dramatically improved.